The long-term goal is the implementation of anti-HIV gene therapy using stem cells as vehicles. The present project targets at the following objectives: 1. To optimize the microenvironment for transduction into stem cells from different sources (bone marrow, umbilical cord and placental blood, peripheral blood upon cytokine stimulations), initially using a reporter gene, neomycin phosphotransferase (neo), and subsequently constructs containing anti-HIV ribozyme genes. 2. To determine the relative merits of different vector systems developed in Project 1 for delivery of anti-HIV ribozymes and other anti- HIV genes into hematopoietic stem cells, such as adeno-associated virus (AAV) vectors, HIV-1(MN) and HIV-2(KR) vectors, or HIV/AAV chimeric vectors. 3. To delineate the differences in subsets of hematopoietic progenitor cells before and after transduction with different vector system by means of multicolor, multidimensional flow cytometric analysis using monoclonal antibodies against a panel of hematopoietic differentiation markers. 4. To assess the stability of expression of the anti-HIV-ribozyme genes by the transduced CD34+ stem cells, their subsets and their progeny cells in short and long-term cultures, as well as by CD34+ cells amplified ex vivo after transduction. 5. Based on the safety and feasibility data from Project 2 and applying the most appropriate vector system and conditions (objectives 1.1. and 1.2.), to develop and explore the clinical use of CD34+ cells derived from umbilical cord and placental blood from newborns of HIV+ mothers for delivery of anti-HIV-ribozyme genes. The following experimental design and methods are used: 1. Multiparameter analysis of the mononuclear cell (MNC) preparations from various sources by multicolor, multiparameter flow cytometry using a panel of monoclonal antibodies against CD3, CD7, CD10, CD11b, CD15, CD33, CD34, CD38, CD45, CD61, CD71, HLA-DR as well as anti-Thy-1. 2. Enrichment of stem cells as defined by presence of CD34 from these various sources by means of an immunomagnetic microsphere method, initially in research scale and subsequently in clinical scale. 3. Establishment of optimal conditions for transduction of neo and subsequently anti-HIV-ribozyme genes using various vector system (AAV, HIV-1, HIV-2, HIV/AAV hybrid), into the CD34+ cells and their subsets. 4. Assessment of stability and expression of the transgenes in the stem cells after short and long-term suspension cultures and subsequently development of the clinical use of CD34+ cells derived from umbilical cord and placental blood for delivery of anti-HIV genes.